Project Summary/Abstract The major objective of this proposal is to test the hypothesis that photoactivation of riboflavin by non- linear optical (NLO), two photon processes (TP) using femtosecond laser (FS) light induces spatially resolved (X-Y-Z) collagen cross-linking (CXL) and mechanical stiffening of intact, live rabbit eyes. Recent studies have established that photoactivation of riboflavin by 370 nm light (UVA) leads to the generation of free radical-induced collagen CXL and corneal stiffening that shows significant therapeutic benefits for patients suffering from Keratoconus. While UVA and riboflavin are themselves non-toxic, the generation of free radicals within the UVA collimated light path can lead to cellular damage affecting the corneal endothelium and crystallin lens, resulting in bullous keratopathy and potentially cataract. To avoid this complication, therapeutic administration of UVA CXL has been titrated to limit free radical generation to the anterior cornea, thus reducing the efficacy of CXL and limiting the therapeutic range to patients with affected corneas thicker than 400 m. We propose that replacement of collimated UVA light with near-infrared FS laser light to induce NLO CXL within a spatially resolved focal volume determined by the focusing lenses would greatly improve the safety and efficacy of CXL. Major benefits of NLO CXL compared to conventional UVA CXL would included: 1) expanded application of corneal CXL to the treatment of thinner ectatic corneas (<400 m) including post LASIK ectasia patients and other corneal disorders currently proposed for conventional UVA CXL including, bullous keratopathy, corneal ulceration and microbial infection; 2) additional treatment areas beyond that of the anterior stroma, including middle, posterior and full thickness corneal stroma; and 3) expanded use of alternative photosensitizers activated by low ultraviolet (UVB and UVC) and visible light (400 to 780 nm) that would be phototoxic to the retina using a single photon approach. To test this hypothesis we propose the following Specific Aims: 1) Adapt the current FS delivery system for live rabbit corneal NLO CXL, 2) Determine the effect of varying TP focal volume, FS laser power and scanning speed on NLO CXL corneal stiffening, 3) Determine the short-term affect (1 and 3 days) of NLO CXL on corneal CAF, 3D corneal elastic modulus, and cell damage compared to UVA CXL, 4) Determine the long-term affects (1 and 6 months) of NLO CXL on corneal CAF, 3D corneal elastic modulus, and cell repair compared to UVA CXL.